p16INK4a expression mediated by recombinant adenovirus can induce senescence of A549 cells / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells.</p><p><b>METHODS</b>p16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells.</p><p><b>RESULTS</b>Immunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells.</p><p><b>CONCLUSION</b>Recombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.</p>